Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.